J. Agric. Food Chem. 1997, 45, 797−802 797

Protein Quality of Concentrates Obtained by Freezing

Teresa Herna´ndez,*,† Carmen Martı´nez,† Amelia Herna´ndez,† and Gloria Urbano‡

Departamento de Nutricio´n y Bromatologı´a, Universidad de Alcala´ de Henares, 28871 Alcala´ de Henares, Madrid, Spain, and Departamento de Fisiologı´a, Universidad de Granada, 18071 Granada, Spain

Freezing protein curd was prepared from alfalfa juice and extracted with 2-propanol, either unwashed or after washing with water. To examine the effect of extraction on digestibility, in vitro digestibility was analyzed for the two extracted protein concentrates and the unextracted protein concentrate. Two methods, one direct, the pH-stat method, and one indirect, the method, were used to determine in vitro digestibility. The protein quality of the unextracted freezing concentrate was also determined by bioassays. The in vivo digestibility of this concentrate (82.8) was lower than that of soybean meal, while the biological value (75.4) was similar. Thus, the lower net protein utilization (62.4) of this concentrate as compared to soybean meal can be ascribed to its lower digestibility. Extraction of the concentrate with 2-propanol lowered in vitro digestibility about 11 and 2% by pH-stat and dietary fiber methods, respectively, while washing the concentrate with water before extraction either had no effect on in vitro digestibility or lowered it slightly. There was an inversely proportional relationship statistically significative between the content of dietary and insoluble fiber in the concentrates and their in vitro digestibilities.

Keywords: Protein quality; protein concentrates; alfalfa

INTRODUCTION 1983). Another method involves extracting whole pro- tein concentrates using organic solvents; this method Unfractionated alfalfa protein concentrates (LPCs) gives higher yields, and the extracted concentrate may have been regarded as a potential alternative to con- be stored for extended periods without need of low ventional sources of edible protein. LPCs have been temperature or exclusion of oxygen (Bray, 1977). assessed as food for children (Kamalanathan et al., 1969, Extraction of the freezing curd with 2-propanol yields 1970; Oke, 1971, 1973; Olatunbosum et al., 1972; concentrates with a high protein content, minimum Kamalanathan and Devadas, 1975; Pirie, 1984) and lipid, sugar, and polyphenol contents, and color and included in food formulations (Toosy and Shah, 1974; texture similar to those of white protein concentrates Meimbam et al., 1982; Lencioni et al., 1984, 1987, 1989; (Herna´ndez et al., 1988, 1991; Herna´ndez and Herna´n- Barbeau and Kinsella, 1987; Maciejewicz-Rys and Hanc- dez, 1994). However, for solvent extraction to be a zakowski, 1990). viable method of preparing chlorophyll-free protein Unfractionated LPC can be prepared by treatment concentrates, it is essential to determine how this using heat (Bickoff et al., 1975), acid (Satake et al., process affects the protein quality of the concentrates 1984), organic solvents (Brown et al., 1975), or polyelec- so produced. trolytes (Anelli et al., 1977; Baraniak et al., 1989). The object of the present study was therefore to Unfractionated LPC can also be obtained by freezing. determine the protein quality of alfalfa protein concen- Freezing alfalfa juice produces a curd, called freezing trates prepared either by extracting freezing curd, curd, which contains 50% of the dry matter and 60% of unwashed or washed with water, with 2-propanol or by the nitrogen present in the original juice (Herna´ndez directly freeze-drying the freezing curd, and hence to et al., 1988). The nitrogen content in the freezing assess the effect of freezing and extraction on the protein concentrate prepared by freeze-drying the curd is the quality of such concentrates. same as in the unfractionated concentrate. This method of preparation offers advantages, in that it requires MATERIALS AND METHODS neither heat nor the addition of any chemical sub- Concentrate Preparation. Preparation of the freezing stances. However, the effect of freezing on the protein curd has been described elsewhere (Herna´ndez et al., 1988, quality of the concentrate so obtained has not yet been 1991). Briefly, alfalfa was harvested, pulped, and pressed. The assessed. juice was poured into small containers and frozen at -25 °C To be suitable for human consumption, LPCs must until use. Each sample was thawed at room temperature for be chlorophyll-free. Such concentrates may be prepared 18 h before use, as needed. Freezing curd so formed was separated from the thawed juice by centrifugation, and some using two different methods. One method is to separate of the freezing curd was freeze-dried to produce freezing the soluble white proteins from the insoluble colored concentrate (FC). Extraction with 2-propanol was carried out protein matter, though this method presents the draw- in a Soxhlet apparatus at the solvent’s boiling point using back of producing lower yields of white protein concen- either untreated curd immediately after preparation, yielding trate (de Fremery et al., 1973; Fiorentini and Galoppini, 2-propanol-extracted freezing concentrate (IFC), or curd after it had been washed with distilled water by centrifugation, yielding water-washed 2-propanol-extracted freezing concen- * Author to whom correspondence should be ad- trate (WIFC) (Figure 1). Following removal of the residual dressed (fax 34-1-8854783). solvent, the extracted FCs were ready for analysis. † Universidad de Alcala´ de Henares. In Vivo Tests of Protein Quality. Apparent (AD) and ‡ Universidad de Granada. true (TD) digestibilities, biological value (BV), and net protein S0021-8561(96)00347-0 CCC: $14.00 © 1997 American Chemical Society 798 J. Agric. Food Chem., Vol. 45, No. 3, 1997 Herna´ndez et al.

Figure 1. Obtention process of freezing protein concentrates. utilization (NPU) of the FC were determined in rats using the 37 °C, and the pH was adjusted to 8.0 and held there for 10 Thomas-Mitchell balance method, as modified by Eggum min. An automatic titration was then run using the pH-stat (1973). The daily weight gain with respect to daily protein procedure, adding 1 mL of the enzyme solution (end point 7.98, intake (DWG/DPI) was calculated for the balance period. 0.100 M NaOH titration solution). Enzyme activity was In experiments, albino Wistar rats (50-60 g) were housed determined on the basis of the amount of 0.100 M NaOH in individual metabolic cages kept in a thermoregulated room required to keep the pH at 7.98 for exactly 10 min. Percentage (21 ( 1 °C) with a controlled 12 h light/dark period. true digestibility (%TD) of the caseinate was calculated by The rats were divided randomly into two groups of 10 animals each one (5 males, 5 females). One group of rats was %TD ) 76.14 + 47.77B used to determine maintenance requirements. The other group was used in the test diet assay. where B ) mL of added 0.100 M NaOH. Values for sodium To determine the maintenance requirements, the animals caseinate digestibility should fall in the range of 98-102% were fed a low-protein (4%) diet of a highly digestible protein (McDonough et al., 1990a). Otherwise, a new three-enzyme (casein + 5% DL-methionine), a sufficient amount to record solution needs to be made up. depletion (McDonough et al., 1990b). To determine the digestibility of the concentrates, an The FC was the only protein source in the test diet at the amount of sample containing 10 mg of N was transferred to a level of 12% of dry matter (DM). The diets also contained 4% titration cell in 10 mL of distilled water. Once the temperature fat, 8% fiber, 1% vitamin mix, and 4% mineral mix, the last had been stabilized at 37 °C, the pH was adjusted to 8 and two according to AIN (1977). A starch and sugar (1:1) mixture held there for 10 min. An amount of 1 mL of the enzyme completed the diets. solution was then added, and the titration was started (end The diets and water were given ad libitum. The rats were point pH 7.98, 0.100 M NaOH titrant solution). The amount fed the low-nitrogen maintenance diet for 6 days (a 3 day of 0.100 M NaOH required to keep the pH at 7.98 for exactly adaptation period and a 3 day balance period) and the test 10 min was used to determine an uncorrected protein digest- diet for 10 days (a 3 day adaptation period and a 7 day balance ibility (%UTD) value by employing the equation set out above. period). At the end of the study the rats were weighed and The %UTD values were corrected using the value obtained the fecal N, urinary N, and food consumed were determined. for the sodium caseinate solution (McDonough et al., 1990a): The AD, TD, BV, and NPU values of the FC were calculated. Moisture was determined by drying at 105 °C and the total %TD ) (%UTD/%TD caseinate) 100 nitrogen according to the Kjeldahl method using a protein × conversion factor of 6.25. The baseline rate of titrant uptake was determined in runs In Vitro Digestibility Method. The in vitro digestibility in which no enzyme solution was added. After the concentrate of the three alfalfa protein concentrates was determined by samples had been adjusted to pH 8.0, the baseline uptake of two methods, a direct assay and an indirect assay. titrant was measured for 10 min. The digestibility values The direct method used was the pH-stat method developed corrected for the baseline consumption of alkali (%TDb) were by Pedersen and Eggum (1983). A three-enzyme solution was obtained. made up to contain 23 100 units of trypsin (EC 3.4.21.4) A Titrino Model SM 702 automatic titrator equipped with (porcine pancreatic trypsin, type IX, Sigma T-0134), 186 units a 10 mL exchange unit (Metrohm) and a thermostated titration of chymotrypsin (EC 3.4.21.1) (bovine pancreatic chymotrypsin, cell was used for all of the pH-stat titrations. type II, Sigma C-4129), and 0.052 unit of peptidase (porcine Protein digestibility was estimated indirectly by determin- intestinal peptidase, grade K, Sigma P-7500) per milliliter. The ing the soluble and insoluble fiber according to the method pH was adjusted to 8.00 at 37 °C and held there for 2 min, developed by Asp et al. (1983), previously applied in the and the enzyme solution was frozen at -30 °C until use. analysis of the fiber in these of concentrates and described in The activity of this solution was determined daily using an detail in Herna´ndez et al. (1995). In that method, following aqueous suspension of sodium caseinate (Sigma C-8654) extraction of the lipids and pigments with (2/1 v/v) chloroform/ containing 1 mg of N/mL of water. An amount of 10 mL of methanol and of the soluble sugars with hot 80% (v/v) ethanol, sodium caseinate suspension was placed in a titration cell at enzymatic hydrolysis of the proteins in the sample was carried Protein Quality of Alfalfa Protein Concentrates J. Agric. Food Chem., Vol. 45, No. 3, 1997 799

Table 1. In Vivo Protein Quality Assessment of the FC (x results were similar to those obtained in an interlabo- ( σn-1, n ) 3) ratory study of the PER organized by AACC/ASTM daily dry daily (Hackler et al., 1984). That study yielded coefficients mean body daily wt, matter intake, protein of variation in the DWG/DPI value of 60.7% for peanut wt, g/rat g/rat g/rat intake, g/rat DWG/DPI meal and 30.1% for a textured vegetable protein at a 64.2 ( 5.6 0.95 ( 0.34 7.4 ( 0.7 0.84 ( 0.08 1.13 ( 0.4 dietary protein content of 10% after an adaptation period of 2 days and a 1-week trial. The coefficients of daily N variation dropped to 22 and 13%, respectively, in trials daily fecal N, mg/rat daily urinary N, mg/rat intake, lasting 3 and 4 weeks. Accordingly, the DWG/DPI value mg/rat total endogenous total endogenous obtained in the present experiment may slightly un- 135.5 ( 13.7 31.4 ( 3.6 8.3 ( 2.7 51.5 ( 7.8 23.3 ( 3.9 derestimate the nutritive value of the FC.

AD TD BV NPU The apparent and true digestibilities of the FC were 76.75 and 82.78%, respectively, as shown in Table 1. 76.7 ( 2.7 82.8 ( 3.1 75.4 ( 8.3 62.4 ( 6.3 The AD value of the FC was similar to that reported for alfalfa whole protein concentrates by Ohshima and out under conditions similar to those used in in vitro methods Ueda (1984) (77%) and by Subba Rau et al. (1972) (78%). of determining digestibility. The hydrolysis employed pepsin (EC 3.4.23.1) in a hydrochloric acid medium for 18 h followed The TD value of the FC was also consistent with the by pancreatin (from hog pancreas) for 1 h, as described by literature values for whole concentrates. Saunders et Schweizer and Wu¨ rsch (1979). The soluble fiber was precipi- al. (1973) reported values of 83-87%, similar to the tated with ethanol out of the supernatant so obtained; the range of 83-89% for whole concentrate recorded by residue comprised the insoluble fiber. The two soluble and Bickoff et al. (1975), who also reported a value of 85% insoluble fiber residues were dried at 105 °C and weighed. The for green concentrate. Subba Rau et al. (1972) reported percentage protein (% N 6.25) remaining in both residues a value of 86% for whole concentrate. × was determined using the Kjeldahl method. Thus, while the AD values obtained by those workers The protein remaining in the soluble and insoluble fiber residues represented the undigestible protein in the sample, were similar to the AD values for the FC in this and those values were used to calculate the protein digest- experiment, the TD values were somewhat higher. This ibility of the concentrates according to discrepancy may be explained by the fact that they, like Saunders et al. (1973), determined the endogenous %D ) 100 - nitrogen in a group of rats fed a protein-free diet, protein (g) in residue protein (g) in residue whereas in this study the rats were fed a diet with a + soluble fiber insoluble fiber low proportion (4%) of a highly digestible protein (casein 100 amount of protein in sample (g) × + 5% DL-methionine), as currently recommended (Mc- Donough et al., 1990a) to avoid excessive loss of organic All determinations of in vitro digestibility were performed by proteins and changes in the intestinal flora. triplicate. Since the digestibility of FC is similar to that of whole and green protein concentrates, the method of preparing RESULTS AND DISCUSSION the FC, by freezing the juice or by means of heat treatment, respectively, could be expected to have little In Vivo Evaluation of the FC. Table 1 presents influence on the in vivo digestibility, dependent mainly the results of the in vivo evaluation of the protein upon the nature of the sample proteins and associated quality of the freezing concentrate. compounds. The daily weight gain with respect to daily protein intake (DWG/DPI) of the FC was 1.13. Subba Rau et Digestibility of the FC can be compared with that for al. (1969) and Myer and Cheeke (1975) recorded similar soybean meals, widely used in the food industry as a PER values of 1.23 and 1.36, respectively, in alfalfa source of protein. The digestibility values found in the whole protein concentrates. Carlsson (1984) reported literature ranged from 85 to 90% (Bickoff et al., 1975; a value of 1.5 for that same type of concentrate. Carlsson and Hanczakowski, 1985; Ohshima and Ueda, Gastineau and de Mathan (1981) recorded a PER value 1984). Consequently, at most the FC is around 7% less of 1.2 for alfalfa green protein concentrate. Bickoff et digestible than soybean meal. al. (1975) obtained corrected PER values of 1.68 for The biological value (BV) and net protein utilization whole protein concentrate and 1.67 for green protein (NPU) of the FC were 75.43 and 62.36%, respectively concentrate. (Table 1). The BV of the FC was the same as that found Although the DWG/DPI value for FC was lower than for whole protein concentrate by Subba Rau et al. (1972). the literature PER values, this may be attributable to Maciejewicz-Rys and Hanczakowski (1990) reported a the shorter duration of the trial in this experiment, 7 BV of 54% for that same type of concentrate, but they days, 3 times shorter than the 28 days of PER trials. In did not explain the method used to prepare the concen- addition, the longer the trial, the more the animals will trate, did not identify the type of rat used, and limited become habituated to the diet, particularly in the case the animals’ daily diet. Furthermore, they also obtained of foods of low palatability, like these concentrates. a very low TD value (65%) as compared with the value Subba Rau et al. (1972) pointed out that acceptance of recorded for FC in the present experiment and the the diet is a limiting factor in methods based on literature values for whole concentrate. In view of the measurements of weight gain. Samonds and Hegsted foregoing, their results would not appear to be compa- (1977) reported that fluctuations in the quantity of food rable. ingested increased the variability in the PER values. Comparing the BV for the FC with the BVs for other In the present trial a coefficient of variation of 10% in food proteins shows it to be similar to the BV for cow’s the amount of DM ingested resulted in a 36% variation milk casein (79.7%) and soybean meal (76.6%) and in the DWG/DPI value. It has not been possible to higher than the BV for isolated soybean protein (66.4%) contrast these values with the values of other workers, (FAO/WHO, 1970). It can thus be concluded that the because they have not been published. However, the proteins in the FC have a high biological value. 800 J. Agric. Food Chem., Vol. 45, No. 3, 1997 Herna´ndez et al.

Table 2. True Digestibility (Percent) of the Alfalfa Pedersen and Eggum (1983) found that the addition Protein Concentrates As Determined by the pH-stat of 5-8 mg of calcium ions increased the digestibility Method (x ( σn-1, n ) 3) values obtained using the pH-stat method by about protein 3-6%. This effect was the reverse when the amount concentrate %TD %TDb added was around 20 mg, and what is more, different FC 93.23 ( 0.36 91.97 ( 0.360 samples responded differently. In addition, Bergmeyer IFC 82.33 ( 0.160 81.79 ( 0.160 (1970) pointed out that calcium ions are activators of WIFC 82.16 ( 0.04 81.41 ( 0.04 trypsin and chymotrypsin. Since calcium concentra- tions in the samples were high, around 5% (DM) Table 3. Digestibility (Pecent) of the Alfalfa Protein (Herna´ndez, 1993), that same effect may have been Concentrates Based on Fiber Dietary Analysis (x ( σn-1, n ) 3) responsible for the high %TD values obtained using the pH-stat method. undigested protein (% DM) The influence of calcium on the in vitro pH-stat protein insoluble soluble total digestibility determination could be confirmed by de- concentrate fiber residue fiber residue fiber residue % D termining digestibility using previously dialyzed samples. FC 7.67 ( 0.01 2.60(0.005 10.3 80.4 Tables 2 and 3 show that the in vitro digestibility IFC 13.37 ( 0.03 1.00 ( 0.005 14.4 78.6 WIFC 14.35 ( 0.04 1.00 ( 0.007 15.4 78.5 values for the extracted concentrates obtained using the pH-stat and pepsin-pancreatin methods were similar. The NPU value for the FC (62.36%) was somewhat Statistical analysis of the digestibility values between lower than the value for whole protein concentrate the two methods indicated that there were significant (66.2%) published by Subba Rau et al. (1972). Com- differences (p < 0.05) and on the order of 2% for the pared to other reference proteins, it was similar to the two concentrates. value for soybean products [meal (60.4 %) and isolated The effect of extraction with 2-propanol and washing protein (63.3%)] and lower than the NPU value for cow’s with water on concentrate digestibility can be inferred milk casein (72.1%) (FAO, 1970). Since the NPU value by comparing the digestibility values for the extracted reflects the BV and digestibility, the fact that the BV concentrates and for the FC determined using the same for the FC was optimal suggests that the lower NPU method. In the case of the pH-stat method, the differ- value must be attributable to the low digestibility of the ence in the %TD values was on the order of 11% for both concentrate. extracted concentrates. Using the pepsin-pancreatin method, the difference was on the order of 2% for both In Vitro Digestibility of the Concentrates. Table of the extracted concentrates. Therefore, extracting the 2 sets out the in vitro digestibility (%TD) values for the freezing curd with 2-propanol appears to decrease the concentrates as determined by the pH-stat method. The digestibility of the proteins in the concentrate, while in vitro digestibility value for the FC was nearly 93%. washing with water before extraction has no effect on That value was around 10% higher than the value the digestibility. determined using bioassays, approximately 83%. The first possible explanation considered was the hydrolyza- The literature contains few and highly disparate data tion of other alkali-consuming substances during the on the effects of extraction of protein concentrates with determination, thus resulting in overestimation of the organic solvents. Experiments have been reported digestibility. To test this hypothesis, alkali consumption using different types of sample, different solvents, and was measured in samples prepared using the same different extraction conditions; what is more, the digest- conditions except that no enzyme was added, and that ibility determinations have also been carried out using baseline consumption value was used to correct the different methods. digestibility determinations. The corrected values (%TDb) Buchanan (1969) determined the effect of heat and are shown in Table 2. The effect of the correction on extraction with chloroform on the digestibility of protein digestibility was very slight, around 1% for the FC and concentrates. Part of a freeze-dried concentrate was even less for the extracted concentrates. On effecting heated in the presence of water in an oven at 105 °C this same correction, Pedersen and Eggum (1983) for 15 h, and part of that heated sample was extracted likewise failed to find variations in the digestibility with chloroform at room temperature. In vivo and in values for samples of different sources. To test whether vitro determinations of digestibility were performed the determination method itself was the cause of the using papain, pepsin, and pepsin-pancreatin. For the discrepancy, the in vitro digestibility was measured determinations using papain, the results showed that using another method based on measurements of the treating the sample with moist heat decreased digest- nitrogen. The procedure for the enzymatic hydrolysis ibility by about 10%, while extraction with chloroform of the proteins used in the analysis of the dietary fiber restored the digestibility to its original value. This was chosen for comparison, because it fulfilled the latter effect was not recorded for the in vivo and in vitro following requirements: the conditions employed were determinations of digestibility carried out using pepsin similar to the conditions used in the in vitro methods and pepsin-pancreatin. of determining digestibility, and undigested nitrogen Savangikar and Ohshima (1987) compared the AD of was analyzed. Accordingly, the undigested protein in leaf protein curds of different plants extracted with the soluble and insoluble fiber residues, expressed as a ethanol without heating but then heated to 70 °C for percentage of the total protein in sample, was calcu- 18 h, with the AD for the same samples freeze-dried or lated, and protein digestibility was then calculated on heated directly, without extraction. On the basis of the basis of those values. The results (table 3) show their results, they concluded that the digestibility of the that the digestibility value for the FC calculated in that treated samples was lower than that of the freeze-dried way was similar to that obtained by the in vivo method. samples and that the decrease was greater in the The presence of a compound acting as an enzyme samples heated directly without extraction (7-10%) activator in the in vitro pH-stat method would provide than in the samples that had been extracted before a possible explanation for that discrepancy. heating (5-7%). Protein Quality of Alfalfa Protein Concentrates J. Agric. Food Chem., Vol. 45, No. 3, 1997 801

Hanczakowski et al. (1991) extracted one part of digestibility values for the different concentrates, ir- alfalfa green protein curd with ethyl ether for 20 h, respective of the method of determination employed. No although they do not say whether the extraction pro- such relationship could be established for the soluble cedure was performed hot or cold. Another part was fiber content, as the results obtained show. washed with water, and yet another part was dried Accordingly, it can be concluded that the digestibility under an infrared lamp at 55 °C. The results indicated of the concentrates decreases as the amount of fiber that extraction with the organic solvent increased increases. The literature contains many reports of this digestibility by 6% and that washing with water had effect, which is ascribable to the ability of fiber to inhibit no effect. various digestive enzymes and to its action as a physical Subba Rau et al. (1972) examined the factors affecting barrier reducing enzyme accessibility (Zebrowska, 1978). leaf protein concentrate quality. They concluded that Heat is another factor that may have exerted an there was an inverse relationship between the ash influence on the lower digestibility of the extracted content, polyphenol content, and soluble solids content concentrates, since a hot extraction process was em- and the nutritive value; they reported that variations ployed. Although heat is beneficial in that it destroys in those factors were manifested mainly in the digest- enzyme inhibitors, it contributes to the formation of ibility value. disulfide bridges and thus to the creation of irreversible The ash contents were similar in the FC and in the steric hindrances to enzymatic action (Richardson and IFC, although it was 2% lower in the WIFC (Herna´ndez, Catsimpoolas, 1979; Hamaker et al., 1987; Serna- 1993), which should have a lower soluble solids content. Saldivar et al., 1988). It will be necessary to verify the Nevertheless, depending upon the method of determi- possible influence of heat by extracting the samples nation applied, the digestibility of the WIFC was the under the same conditions at room temperature. same or lower than that of the IFC and substantially lower than that of the FC. This finding agrees with the ACKNOWLEDGMENT results, already discussed above, reported by Hancza- We thank the Instituto de Nutricio´n y Tecnologı´a de kowski et al. (1991), who also found no improvement Alimentos at the University of Granada, Spain, for on washing the concentrate with water. It disagrees technical assistance and Mr. Russell Sacks for linguistic with the results of Subba Rau et al. (1972), who found assistance. that a lower ash content did improve digestibility. Extraction lowers the amount of polyphenols in the LITERATURE CITED concentrate, and washing the sample with water results in an even larger decrease (Herna´ndez et al., 1991). AIN ad Hoc Committe on Standards for Nutritional Studies. Consequently, the experimental results showed no Report of the AIN ad Hoc Committee an Standards for correlation between polyphenols and digestibility. This Nutritional Studies. J. Nutr. 1977, 107, 1340-1348. agrees with the findings obtained by Hanczakowski et Anelli, G.; Fiorentini, R.; Massignan, L.; Galoppini, C. The al. (1991), who prepared an alfalfa green protein con- polyprotein process: a new method for obtaining leaf protein centrate with a low polyphenol content by washing the concentrates J. Food Sci. 1977, 42, 1401-1403. Asp, N. G.; Johansson, C. G.; Hallmer, H.; Siljestro¨m, M. Rapid curd with a 40 mM sodium phosphate buffer solution enzymatic assay of insoluble and soluble dietary fiber. J. (pH 7) containing 2.5% NaCl and 1 mM 2-mercapto- Agric. Food Chem. 1983, 31, 476-482. ethanol without obtaining any improvement in digest- Baraniak, B.; Baraniak, A.; Bubicz, M. Protein concentrate ibility. That same washing solution was used by Lahiry from alfalfa and cocksfoot by polyelectrolyte precipitation. et al. (1977) to reduce the chlorogenic acid content. They Nahrung 1989, 33, 491-495. reported a 1.8% increase in the in vitro digestibility of Barbeau, W. E.; Kinsella, J. E. Formation of a gel from a whole alfalfa concentrate as determined using the heated emulsion of alfalfa leaf protein and peanut oil. J. method of Hsu et al. (1977), and they attributed this Food Sci. 1987, 52, 1030-1032. finding to possible inhibition of proteolytic enzymes by Bergmeyer, H. V. Methods of Enzymatic Analysis, 2nd ed.; the polyphenols. Verlag Chemie, Weinheim, Germany, 1970. Bickoff, E. M.; Booth, A. N.; de Fremery, D.; Edwards, R. H.; The absence of any correlation between digestibility Knuckles, B. E.; Miller, R. E.; Saunders, R. M.; Kohler, G. and the contents of these factors in the concentrates in O. Nutritional evaluation of alfalfa leaf protein concentrate. this experiment suggests that the ash, soluble solids, In Protein Nutritional Quality of Foods and Feeds; Fried- and polyphenol contents have no influence on their man, M., Ed.; Dekker: New York, 1975; Vol. 2, Chapter 13. protein digestibility. Bray, W. J. The processing of leaf proteins to obtain food-grade However, comparison between the dietary fiber con- products. In Green Crop Fractionation; Symposium 9; tent of the concentrates and their digestibility values Wilkins, R. J., Ed.; British Grassland Society: Hurley, U.K., reveals an inversely proportional relationship. Correla- 1977. Brown, H. E.; Stein, E. R.; Saldana, G. Evaluation of Brassica tion analyses were run on the insoluble, soluble, and carinata as a source of plant protein. J. Agric. Food Chem. total dietary fiber contents (Herna´ndez et al., 1995) and 1975, 23, 545-547. on the corresponding digestibility values for the con- Buchanan, R. A. In vivo and in vitro methods of measuring centrates as determined by in vitro methods. nutritive value of leaf protein preparations. Br. J. Nutr. The correlation coefficient values obtained using the 1969, 23, 533-545. pH-stat and pepsin-pancreatin methods were, respec- Carlsson, R. Effects of species, plant physiological development tively, -0.9649 (n ) 2, p ) 0.1690) and -0.9754 (n ) 2, stages, growth conditions, and processing conditions on the p ) 0.1414) for the insoluble fiber content, -0.8663 (n nutritive value of leaf protein concentrates. Curr. Trends 2, p 0.8416) and 0.8438 (n 2, p 0.0213) for Life Sci. 1984, 11 (Progress in Leaf Protein Research), 201- ) ) - ) ) 208. the soluble fiber content, and -0.9967 (n ) 2, p ) Carlsson, R.; Hanczakowski, P. The nutritive value of mixtures 0.0512) and -0.9993 (n ) 2, p ) 0.0236) for the total of white leaf protein and food proteins. J. Sci. Food Agric. fiber content. These results are suggestive of a highly 1985, 36, 946-950. significant linear relationship between the insoluble de Fremery, D.; Miller, R. E.; Edwards, R. H.; Knuckles, B. fiber content, and especially the total fiber content, and E.; Bickoff, E. M.; Kohler, G. O. Centrifugal separation of 802 J. Agric. Food Chem., Vol. 45, No. 3, 1997 Herna´ndez et al.

white and green protein fractions from alfalfa juice following McDonough, F. E.; Sarwar, G.; Steinke, F. H.; Slump, P.; controlled heating. J. Agric. Food Chem. 1973, 25, 886- Garcı´a, S.; Boisen, S. In vitro assay for protein digestibility: 889. interlaboratory study. J. Assoc. Off. Anal. Chem. 1990a, 73, Eggum, B. O. A study of certain factors influencing protein 622-625. utilization in rats and pigs. 406. beretn. Thesis, The McDonough, F. E.; Steinke, F. H.; Sarwar, G.; Eggum, B. O.; National Institute of Animal Science, Copenhagen, 1973. Bressani, R.; Huth, P. J.; Barbeau, W. E.; Mitchell, G. V.; FAO. Content of Foods and Biological Data on Phillips, J. G. In vivo rat assay for the protein digestibility: Proteins; FAO: Rome, 1970. collaborative study. J. Assoc. Off. Anal. Chem. 1990b, 73, Fiorentini, R.; Galoppini, C. The proteins from . Qual. 801-805. Plant.-Plant Foods Hum. Nutr. 1983, 32, 335-350. Meimbam, E. J.; Bautista, J. G.; Soriano, M. R. Studies on Gastineau, I.; de Mathan, O. La pre´paration industrielle de the fortification of biscuits with leaf protein con- la prote´ine verte de luzerne. In Proteines Foliares et Ali- centrates. Philipp. J. Nutr. 1982, 35,82-86. mentation; Costes, C., Ed.; Gauthier-Villars: Paris, 1981. Myer, R. O.; Cheeke, P. R. Utilization of alfalfa meal and Hackler, L. R.; Bodwell, C. E.; Happich, M. L.; Phillips, J. G.; alfalfa protein concentrate by rats. J. Anim. Sci. 1975, 40, Derse, P. H.; Elliott, J. G.; Hartnagel, R. E.; Hopkins, D. 500-508. T.; Kapiszka, E. L.; Mitchell, G. V.; Parsons, G. F.; Prescher, Ohshima, M.; Ueda, H. Effects of some treatments on the yield E. E.; Robaidek, E. S.; Womack, M. Protein efficiency ratio: and the nutritive value of lucerne leaf protein concentrate. AACC/ASTM collaborative study. J. Assoc. Off. Anal. Chem. Jpn. J. Zootech. Sci. 1984, 55, 584-590. 1984, 67,66-77. Oke, O. L. Some aspects of leaf protein work in Nigeria. Indian Hamaker, B. R.; Kirleis, A. W.; Butler, L. G.; Axtell, J. D.; J. Nutr. Diet. 1971, 8, 121-129. Mertz, E. T. Improving the in vitro protein digetibility of Oke, O. L. Leaf protein research in Nigeria: a review. Trop. sorghum with reducing agents. Proc. Natl. Acad. Sci. U.S.A. Sci. 1973, 15, 139. 1987, 84, 626-628. Olatunbosum, D. A.; Adadevoch, E. K.; Oke, O. L. A new source Hanczakowski, P.; Szymczyk, B.; Skraba, B. Composition and for the management of protein calorie malnutrition in nutritive value of native and modified green fraction of leaf Nigeria. Niger. Med. J. 1972, 2, 195-199. protein from lucerne (Medicago sativa). J. Sci. Food Agric. Pedersen, B.; Eggum, B. O. Prediction of protein digestibility 1991, 56, 495-501. by in vitro enzymatic pH-stat procedure. Z. Tierphysiol., Herna´ndez, T. Obtencio´n y evaluacio´n nutricional de concen- Tierernaehr. Futtermittelkd. 1983, 49, 265-277. trados proteicos foliares de alfalfa (Medicago sativa). Ph.D. Pirie, N. W. Factors affecting beta-carotene destruction in Thesis, Universidad de Alcala´ de Henares, 1993. moist preserved leaf protein. Qual. Plant.-Plant Foods Hum. Herna´ndez, T.; Herna´ndez, A. Available carbohydrates in leaf Nutr. 1984, 34, 229-237. protein concentrates. J. Agric. Food Chem. 1994, 42, 1747- Richardson, D. P.; Catsimpoolas, N. The effect of thermal 1749. denaturation on the tryptic hydrolysis of glycinin. J. Sci. Herna´ndez, A.; Martı´nez, C.; Gonza´lez, G. Freezing of alfalfa Food Agric. 1979, 30, 463-468. leaf juice. Formation and solvent extraction of freezing curd. Samonds, K. W.; Hegsted, D. M. Animal bioassays: a critical J. Sci. Food Agric. 1988, 42, 173-182. evaluation with specific reference to assessing nutritive Herna´ndez, T.; Herna´ndez, A.; Martı´nez, C. Polyphenols in value for the human. In Evaluation of Protein for Humans; alfalfa leaf concentrates. J. Agric. Food Chem. 1991, 39, Bodwell, C. E., Ed.; Avi Publishing: Westport, CT, 1977; 1120-1122. Chapter 4. Herna´ndez, T.; Martı´nez, C.; Herna´ndez, A. Influence of Satake, I.; Sugiura, M.; Kubota, T.; Kobayashi, T.; Sasaki, T.; solvent extraction on the dietary fiber fractions of alfalfa Makino, S.; Kamaya, Y. Clover LPC decolorized by ethanol protein concentrates. J. Sci. Food Agric. 1995, 68, 187-190. extraction. Nippon Shokuhin Kogyo Gakkaishi 1984, 31, Hsu, H. W.; Vavak, D. L.; Satterlee, L. D.; Miller, G. A. A 161-167. multienzyme technique for estimating protein digestibility. Saunders, R. M.; Connor, M. A.; Booth, A. N.; Bickoff, E. M.; J. Food Sci. 1977, 42, 1269-1273. Kohler, G. O. Measurement of digestibility of alfalfa protein Kamalanathan, G.; Devadas, R. P. Leaf protein as supplement concentrates by in vivo and in vitro methods. J. Nutr. 1973, in preschool feeding programs. Presented at the 10th 103, 530-534. International Congress on Nutrition, Kyoto, 1975. Savangikar, C.; Ohshima, M. Application of in vitro methods Kamalanathan, G.; Usha, M. S.; Devadas, R. P. Evolution of to assess the nutritive value of leaf protein concentrates. J. acceptability of some recipes with leaf protein concentrates. Agric. Food Chem. 1987, 35,82-85. Indian J. Nutr. Diet. 1969, 6,12-44. Schweizer, T. F.; Wu¨ rsch, P. Analysis of dietary fibre. J. Sci. Kamalanathan, G.; Nalinakshi, G. S.; Devadas, R. P. Effect Food Agric. 1979, 30, 613-619. of a blend of protein foods on the nutritional status of Serna-Saldivar, S. O.; Tellez-Giron, A.; Rooney, L. W. Produc- preschool children in a rural blawadi. Indian J. Nutr. Diet. tion of tortilla chips from sorghum and maize. J. Cereal Sci. 1970, 7, 288-291. 1988, 8, 257-284. Lahiry, N. L.; Satterlee, L. D.; Hsu, H. W.; Wallace, G. W. Subba Rau, B. H.; Mahadeviah, S.; Singh, N. Nutritional Characterization of chlorogenic acid binding fraction in leaf studies on whole-extract coagulated leaf protein and frac- protein concentration. J. Food Sci. 1977, 42,83-85. tionated chloroplastic and cytoplasmic proteins from lucerne Lencioni, L.; Fiorentini, R.; Galoppini, C. Caseificazione di latte (Medicago sativa). J. Sci. Food Agric. 1969, 20, 355-358. di pecora miscelato con succo di erba medica. Ind. Aliment. Subba Rau, B. H.; Ramana, K. V.; Johnson, C. B. Studies on 1984, 23, 106-111. nutritive value of leaf proteins and some factors affecting Lencioni, L.; Pisanelli, A. M.; Baldi, E.; Fiorentini, R.; Galop- their quality. J. Sci. Food Agric. 1972, 23, 233-245. pini, C. Sheep milk cheese made with the addition of alfalfa Toosy, R. Z.; Shah, F. N. Leaf protein concentrate in human leaf protein concentrate. Proteolysis during ripening. Leb- diet. J. Sci. Ind. Res. 1974, 17,40-42. ensm. Wiss. Technol. 1987, 20, 277-281. Zebrowska, T. Determination of available amino acids in Lencioni, L.; Pisanelli, A. M.; Baldi, E.; Fiorentini, R.; Galop- feedstuffs for monogastrics. Feedstuffs 1978, 50,43-44. pini, C. Sheep milk cheese made with the addition of alfalfa leaf protein concentrate. Acidity and texture during ripen- ing. Lebensm. Wiss. Technol. 1989, 22,81-87. Received for review May 8, 1996. Revised manuscript received X Maciejewicz-Rys, J.; Hanczakowski, P. Improvement of the October 30, 1996. Accepted November 25, 1996. nutritive value of cereals by leaf protein supplemtation. J. JF960347E Sci. Food Agric. 1990, 50,99-104. Maliwal, P. B. In vitro methods to assess the nutritive value of leaf protein concentrate. J. Agric. Food Chem. 1983, 31, X Abstract published in Advance ACS Abstracts, Janu- 315-319. ary 15, 1997.